Interaction of CPa-1 with the manganese-stabilizing protein of photosystem II: identification of domains cross-linked by 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide.

The structural organization of photosystem II proteins has been investigated by use of the zero-length protein cross-linking reagent 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide and monoclonal and polyclonal antibody reagents. Photosystem II membranes were treated with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide which cross-links amino groups to carboxyl groups which are in van der Waals contact. This treatment did not affect the oxygen evolution rates of these membranes and increased the retention of oxygen evolution after CaCl2 washing. Analysis of the proteins cross-linked by this treatment indicated that two cross-linked species with apparent molecular masses of 95 and 110 kDa were formed which cross-reacted with antibodies against both the 33-kDa manganese-stabilizing protein and the chlorophyll protein CPa-1. Cleavage of the 110-kDa cross-linked species with cyanogen bromide followed by N-terminal sequence analysis was used to identify the peptide fragments of CPa-1 and the manganese-stabilizing protein which were cross-linked. Two cyanogen bromide fragments were identified with apparent molecular masses of 50 and 25 kDa. N-Terminal sequence analysis of the 50-kDa cyanogen bromide fragment indicates that this consists of the C-terminal 16.7-kDa fragment of CPa-1 and the intact manganese-stabilizing protein. This strongly suggests that the manganese-stabilizing protein is cross-linked to the large extrinsic loop domain of CPa-1. N-Terminal analysis of the 25-kDa cyanogen bromide fragment indicates that this consists of the C-terminal 16.7-kDa peptide of CPa-1 and the N-terminal 8-kDa peptide of the manganese-stabilizing protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of CPa-1 with the manganese-stabilizing protein of photosystem II: identification of domains on CPa-1 which are shielded from N-hydroxysuccinimide biotinylation by the manganese-stabilizing protein.

The structural organization of photosystem II proteins has been investigated by use of the amino group-labeling reagent N-hydroxysuccinimidobiotin (NHS-biotin) and calcium chloride-washed photosystem II membranes. We have previously shown that the presence of the extrinsic, manganese-stabilizing protein on photosystem II membranes prevents the modification of lysyl residues located on the chlorophyll protein CPa-1 (CP-47) by NHS-biotin [Bricker, T. M., Odom, W. R., & Queirolo, C. B. (1988) FEBS Lett. 231, 111-117]. Upon removal of the manganese-stabilizing protein by calcium chloride-washing, CPa-1 can be specifically modified by treatment with NHS-biotin. Preparative quantities of biotinylated CPa-1 were subjected to chemical cleavage with cyanogen bromide. Two major biotinylated peptides were identified with apparent molecular masses of 11.8 and 15.7 kDa. N-terminal sequence analysis of these peptides indicated that the 11.8-kDa peptide was 232G-330M and that the 15.7-kDa peptide was 360P-508V. The 15.7-kDa CNBr peptide was subjected to limited tryptic digestion. The two smallest tryptic fragments identified migrated at apparent molecular masses of 9.1 (nonbiotinylated) and 7.5 kDa (biotinylated). N-terminal sequence analysis and examination of the predicted amino acid sequences of these peptides suggest that the 9.1-kDa fragment was 422R-508V and that the 7.5-kDa fragment was 360P-421A. These results strongly suggest that two NHS-biotinylated domains, 304K-321K and 389K-419K, become exposed on CPa-1 when the manganese-stabilizing protein is removed by CaCl2 treatment. Both of these domains lie in the large extrinsic loop E of CPa-1.     

Rye photosystem II. Nucleotide sequence of the psbC gene coding 43- kDa chlorophyll(a)-binding proteins

The structure of the rye chloroplast DNA, which contains psbC gene coding for 43-kDa chlorophyll(a)-binding subunit of photosystem II, is determined. The sequence of trnS (UGA) gene encoding tRNA Ser is located at a distance of 140 bp downstream from the stop codon of psbC gene on the opposite DNA strand. The 5'-terminal part of psbC gene, like in other plants, overlaps by 50 bp the 3'-terminal region of psbD gene coding for D2 protein of photosystem II. The amino acid sequence of the psbC gene product reveals common features with the structure of the psbB gene product (CPa-1 protein). The structural similarity of these two proteins seems to reflect their similar functions.     

Characterization of a spinach photosystem II core preparation isolated by a simplified method

A photosystem II core complex from spinach exhibiting high rates of electron transport was obtained rapidly and in high yield by treatment of a Tris-extracted, O2-evolving photosystem II preparation with the detergent dodecyl-beta-D-maltoside. The core complex was essentially free of light-harvesting chlorophyll-protein and photosystem I polypeptides, and was highly enriched in the polypeptides associated with the photosystem II reaction center (45 and 49 kDa), cytochrome b559, and three polypeptides in the region 32-34 kDa. The photosystem II core complex contained two chlorophyll-proteins which had a slightly higher apparent molecular mass than CPa-1 and CPa-2. Additionally, a high-molecular-mass chlorophyll-protein complex termed CPa* was observed, which exhibited a low fluorescence yield when illuminated with ultraviolet light. This observation suggests that CPa* contains a functionally efficient quencher of chlorophyll fluorescence, possibly P680.     

Quality control of photosystem II

Photosystem II is particularly vulnerable to excess light. When illuminated with strong visible light, the reaction center D1 protein is damaged by reactive oxygen molecules or by endogenous cationic radicals generated by photochemical reactions, which is followed by proteolytic degradation of the damaged D1 protein. Homologs of prokaryotic proteases, such as ClpP, FtsH and DegP, have been identified in chloroplasts, and participation of the thylakoid-bound FtsH in the secondary degradation steps of the photodamaged D1 protein has been suggested. We found that cross-linking of the D1 protein with the D2 protein, the alpha-subunit of cytochrome b(559), and the antenna chlorophyll-binding protein CP43, occurs in parallel with the degradation of the D1 protein during the illumination of intact chloroplasts, thylakoids and photosystem II-enriched membranes. The cross-linked products are then digested by a stromal protease(s). These results indicate that the degradation of the photodamaged D1 protein proceeds through membrane-bound proteases and stromal proteases. Moreover, a 33-kDa subunit of oxygen-evolving complex (OEC), bound to the lumen side of photosystem II, regulates the formation of the cross-linked products of the D1 protein in donor-side photoinhibition of photosystem II. Thus, various proteases and protein components in different compartments in chloroplasts are implicated in the efficient turnover of the D1 protein, thus contributing to the control of the quality of photosystem II under light stress conditions.     

Isolation of PS II reaction centre and its relationship to the minor chlorophyll-protein complexes

Evidence is presented for the identification of the chlorophyll- protein complex CPa-1 (CP 47) as the reaction centre of photosystem II (PS II). We have developed a simple, rapid method using octyl glucoside solubilization to obtain preparations from spinach and barley that are highly enriched in PS II reaction centre activity (measured as the light-driven reduction of diphenylcarbazide by 2,6-dichlorophenolindophenol). These preparations contain only the two minor chlorophyll-protein complexes CPa-1 and CPa-2. During centrifugation on a sucrose density gradient, there is a partial separation of the two CPa complexes from each other, and a complete separation from other chlorophyll-protein complexes. The PS II activity comigrates with CPa-1 but not CPa-2, strongly suggesting that the former is the reaction centre complex of PS II. Reaction centre preparations are sensitive to the herbicide 3(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), but only at much higher concentrations than those required to inhibit intact thylakoid membranes. A model of PS II incorporating our current knowledge of the chlorophyll-protein complexes is presented. It is proposed that CPa-2 and the chlorophyll a + b complex CP 29 may function as internal antenna complexes surrounding the reaction centre, with the addition of variable amounts of the major chlorophyll a + b light-harvesting complex.     

In vitro synthesis and assembly of photosystem II proteins of spinach chloroplasts

The synthesis and assembly of photosystem II (PS II) proteins of spinach chloroplasts were investigated in three different in vitro systems, i.e., protein synthesis in isolated chloroplasts (in organello translation), read-out translation of thylakoid-bound ribosomes, and transport of translation products from spinach leaf polyadenylated RNA into isolated chloroplasts. Polyacrylamide gel electrophoresis of labeled thylakoid polypeptides in the presence of sodium dodecyl sulfate revealed that the first two systems were capable of synthesizing the reaction center proteins of PS II (47 and 43 kDa), the herbicide-binding protein, and cytochrome b559. The reaction center proteins synthesized in organello were shown to bind chlorophyll and to assemble properly into the PS II core complex. One of the reaction center proteins translated by the thylakoid-bound ribosomes (47 kDa) was also found to be integrated in situ into the complex but was lacking bound chlorophyll. Incorporation of radioactivity into the three extrinsic proteins of the oxygen-evolution system (33, 24, and 18 kDa) was detected only when intact chloroplasts were incubated with the translation products from polyadenylated RNA, showing that these proteins are coded for by nuclear DNA. The occurrence of a precursor polypeptide 6 kDa larger than the 33-kDa protein was immunochemically detected in the translation products.     

Characterization of a full-length petunia cDNA encoding a polypeptide of the light-harvesting complex associated with photosystem I

We have isolated and characterized a full-length cDNA clone (LHCI-15) which specifies a new chlorophyll-binding protein. This protein is associated with the light-harvesting complex of photosystem I (LHCI). The DNA sequence predicts a precursor protein of 270 amino acids, which shares significant homology with the amino acid sequence of another chlorophyll-binding protein; the chlorophyll a/b-binding (Cab) protein of the photosystem II light-harvesting complex (LHCII). There are two extensive regions of homology (at least 45 residues each) which have approximately 50% amino acid sequence identity. These regions coincide with two of the proposed membrane-spanning alpha helices in the Cab proteins of the LHCII and probably include conserved chlorophyll-binding sites. The LHCI-15 cDNA hybridizes to at least 7 genomic EcoRI DNA fragments, which are very closely related at the nucleotide sequence level.     

Importance of the cyanobacterial Gun4 protein for chlorophyll metabolism and assembly of photosynthetic complexes

Gun4 is a porphyrin-binding protein that activates magnesium chelatase, a multimeric enzyme catalyzing the first committed step in chlorophyll biosynthesis. In plants, GUN4 has been implicated in plastid-to-nucleus retrograde signaling processes that coordinate both photosystem II and photosystem I nuclear gene expression with chloroplast function. In this work we present the functional analysis of Gun4 from the cyanobacterium Synechocystis sp. PCC 6803. Affinity co-purification of the FLAG-tagged Gun4 with the ChlH subunit of the magnesium chelatase confirmed the association of Gun4 with the enzyme in cyanobacteria. Inactivation of the gun4 gene abolished photoautotrophic growth of the resulting gun4 mutant strain that exhibited a decreased activity of magnesium chelatase. Consequently, the cellular content of chlorophyll-binding proteins was highly inadequate, especially that of proteins of photosystem II. Immunoblot analyses, blue native polyacrylamide gel electrophoresis, and radiolabeling of the membrane protein complexes suggested that the availability of the photosystem II antenna protein CP47 is a limiting factor for the photosystem II assembly in the gun4 mutant.     

Chlorophyll-proteins from maize seedlings grown under intermittent light conditions

We studied the organization of the antenna system of maize (Zea mays L.) seedlings grown under intermittent light conditions for 11 d. These plants had a higher chlorophyll-a/b ratio, a higher ratio of carotenoids to chlorophyll and a lower ratio of chlorophyll to protein than plants grown in continuous light. We found all chlorophyll-protein complexes of maize to be present. However, the minor chlorophyll a/b-proteins CP29 and CP26, and to a greater extent CP24 and the major light-harvesting complex II were reduced relative to the photosystem (PS) II core-complex. Also the chlorophyll a/b-antennae of PSI were reduced relative to the reaction-centre polypeptides. When isolated by flatbed isoelectrofocussing, the chlorophyll-a/b complexes of PSII showed a higher chlorophyll-a/b ratio and a lower ratio of chlorophyll to protein than the same complexes from continuous light; additionally, they bound more carotenoids per protein than the latter. Thus the altered organization of the photosynthetic apparatus of plants from intermittent light is caused by two different factors: (i) the altered stoichiometry of chlorophyll-binding proteins and (ii) a different ratio of pigment to protein within individual chlorophyll-proteins.Abbreviations Chl chlorophyll - CL continuous light - F fraction - HPLC high-performance liquid chromatography - IEF isoelectrofocussing - IL intermittent light - LHCII light-harvesting complex II - PAGE polyacrylamide-gel electrophoresis - Phe pheophytin - SDS sodium dodecyl sulfate This work was supported by the grant no. 4.7240.90 from the Italian Ministry of Agriculture and Forestry. We thank Drs. R. Barbato (Dipartimento di Biologia, Padua, Italy) and Olivier Vallon (Institut de Biologie Physico-Chimique, Paris, France) for their gifts of antibodies, Drs. R. Barbato and P. Dainese (Dipartimento di Biologia, Padua, Italy) for fruitful discussion and Prof. G. Gennari (Dipartimento di Chimica fisica, Padua, Italy) for his assistance in recording the excitation spectra. J.M. was supported by a Stipendium from the Deutsche Forschungsgemeinschaft, which is gratefully acknowledged.     

Leaf senescence in a non-yellowing mutant of Festuca pratensis: Proteins of photosystem II

The senescence of leaves is characterized by yellowing as chlorophyll pigments are degraded. Proteins of the chloroplasts also decline during this phase of development. There exists a non-yellowing mutant genotype of Festuca pratensis Huds. which does not suffer a loss of chlorophyll during senescence. The fate of chloroplast membrane proteins was studied in mutant and wild-type plants by immune blotting and immuno-electron microscopy. Intrinsic proteins of photosystem II, exemplified by the light-harvesting chlorophyll a/b-binding protein (LHCP-2) and D1, were shown to be unusually stable in the mutant during senescence, whereas the extrinsic 33-kilodalton protein of the oxygen-evolving complex was equally lable in both genotypes. An ultrastructural study revealed that while the intrinsic proteins remained in the internal membranes of the chloroplasts, they ceased to display the heterogenous lateral distribution within the lamellae which was characteristic of nonsenescent chloroplasts. These observations are discussed in the light of possible mechanisms of protein turnover in chloroplasts.Abbreviations kDa kilodalton - LHCP-2 light-harvesting chlorophyll a/b-binding protein - M_r relative molecular mass - PSII photosystem II - SDS sodium dodecyl sulphate     

Spectral Properties and Composition of Reaction Center and Ancillary Polypeptide Complexes of Photosystem II Deficient Mutants of Synechocystis 6803

The polypeptide composition and spectral properties of three photosystem II (PSII) deficient mutants of the cyanobacterium Synechocystis 6803 have been determined. The levels of the 43 and 47 kilodalton chlorophyll-binding proteins and the reaction center component D2 are affected differently in each mutant; the 33 kD polypeptide of the oxygen-evolving complex is found at wild-type levels in all three. The 43 and 47 kilodalton proteins are implicated as important elements in the assembly and/or stability of the PSII reaction center, although the loss of one of these polypeptides does not lead to the loss of all PSII proteins. Low temperature fluorescence emission spectra of wild-type cells reveal chlorophyll-attributable peaks at 687 (PSII), 696 (PSII), and 725 (photosystem I) nanometers. All three mutants retain the 725 nanometer fluorescence but lack the 696 nanometer peak. This suggests that the latter fluorescence arises from PSII reaction center chlorophyll or results from interactions among functional PSII components in vivo. Cells that contain the 43 kilodalton and lack the 47 kilodalton protein, retain the 687 fluorescence; furthermore, in as much as this fluorescence is absent from cells without the 43 kilodalton protein, the 687 nanometer peak is judged to emanate from the 43 kilodalton chlorophyll-protein. A new peak, probably previously obscured, is revealed at 691 nanometers in cells that retain the 47 kilodalton protein but lack the 43 kilodalton polypeptide, suggesting that emission near 691 nanometers can be attributed to the 47 kilodalton polypeptide. Membrane-bound phycobilisomes are retained in these cells as is coupled-energy transfer between phycocyanin and allophycocyanin. Energy transfer to photosystem I by way of phycocyanin excitation proceeds as in wild-type cells despite the absence of certain PSII components.     

Functional discrimination between Photosystem-II associated chlorophyll a proteins in Zea mays

Three minor Chl a proteins were detected in electrophoretic profiles from wild-type maize thylakoids. The spectral characteristics of these Chl proteins and the apparent molecular weights of their constituent apoproteins suggested that they were associated with the Photosystem-II reaction center. One of these Chl a-proteins, CPa-1, was present in wild-type thylakoids and a photochemically active Photosystem-II particle, but was missing from thylakoids of a mutant-lacking Photosystem-II reaction center. CPa-2, on the other hand, was enriched in mutant thylakoids but was completely missing from the Photosystem-II particles. We conclude that CPa-1 is most likely to contain the photoactive chlorophyll of Photosystem II, while CPa-2 is not required for Photosystem-II activity. The apparent molecular weights of the major CPa-1 and CPa-2 apoproteins were 48 000 and 42 000, respectively. The third minor Chl protein seems most likely to be an electrophoretic variant of CPa-1 and has been designated CPa-11. Seven other Chl proteins were detected in wild-type profiles. Many of these Chl proteins appeared to be oligomers or highly order complexes of LHCP and CP-1.     

Chlorophyll-Protein Complexes of a Photosystem II Mutant of Maize : Evidence that Chlorophyll-Protein a-2 and a Chlorophyll-Protein Complex Derived from a Photosystem I Antennae System Comigrate on Polyacrylamide Gels

Use of the octyl β-d-glucopyranoside solubilization procedure of Camm and Green (1980 Plant Physiol 66: 428-432) reveals that thylakoid membranes of a photosystem (PS) II-deficient maize (Zea mays L.) mutant lack two chlorophyll protein (CP) complexes associated with PSII, i.e. CPa-1 and CPa-2. In contrast, when lithium dodecyl sulfate is used to solubilize the membranes of the mutant prior to electrophoretic separation, a CP complex is observed which has a mobility similar to that of CPa-2. Comparison of spectral characteristics and polypeptide composition of the green bands in this region taken from samples of the mutant, normal sibling control plants and from PSII preparations indicate that the CP complex observed in the mutant represents a portion of a light-harvesting complex of PSI (Mullet et al. 1980 Plant Physiol 65: 814-822). The green band observed in normal maize samples can contain both the CPa-2 complex as well as the CP complex derived from the PSI antennae system.     

Chlorophyll-proteins of the photosystem II antenna system

The chlorophyll-protein complexes of purified maize photosystem II membranes were separated by a new mild gel electrophoresis system under conditions which maintained all of the major chlorophyll a/b-protein complex (LHCII) in the oligomeric form. This enabled the resolution of three chlorophyll a/b-proteins in the 26-31-kDa region which are normally obscured by monomeric LHCII. All chlorophyll a/b-proteins had unique polypeptide compositions and characteristic spectral properties. One of them (CP26) has not previously been described, and another (CP24) appeared to be identical to the connecting antenna of photosystem I (LHCI-680). Both CP24 and CP29 from maize had at least one epitope in common with the light-harvesting antennae of photosystem I, as shown by cross-reactivity with a monoclonal antibody raised against LHCI from barley thylakoids. A complex designated Chla.P2, which was capable of electron transport from diphenylcarbazide to 2,6-dichlorophenolindophenol, was isolated by nondenaturing gel electrophoresis. It lacked CP43, which therefore can be excluded as an essential component of the photosystem II reaction center core. Fractionation of octyl glucoside-solubilized photosystem II membranes in the presence and absence of Mg2+ enabled the isolation of the Chla . P2 complex and revealed the existence of a light-harvesting complex consisting of CP29, CP26, and CP24. This complex and the major light-harvesting system (LHCII) are postulated to transfer excitation energy independently to the photosystem II reaction center via CP43.     

Isolation of a manganese-containing protein complex from photosystem II preparations of spinach

Purified 125I-labeled 33-kDa protein binds to calcium-washed photosystem II preparations at high-affinity and low-affinity binding sites. Filling 70% of the high-affinity site with 33-kDa protein induces 63% of the maximum achievable reconstitution of O2-evolving activity. When N-succinimidyl [(4-azidophenyl)dithio]propionate modified 33-kDa protein was reconstituted into Ca(II)-washed membranes under conditions that primarily filled the high-affinity site and then cross-linked to adjacent proteins by illumination of the photoaffinity label, a cross-linked protein complex was formed that could be solubilized from the membranes with sodium dodecyl sulfate. The protein complex consisted of 22-, 24-, 26-, 28-, 29-, and 31-kDa proteins cross-linked to the 33-kDa protein and contained about 3-4 mol of Mn/mol of protein.     

The identification of IsiA proteins binding chlorophyll <Emphasis Type="Italic">d</Emphasis> in the cyanobacterium <Emphasis Type="Italic">Acaryochloris marina</Emphasis>

The bioavailable iron in many aquatic ecosystems is extremely low, and limits the growth and photosynthetic activity of phytoplankton. In response to iron limitation, a group of chlorophyll-binding proteins known as iron stress-induced proteins are induced and serve as accessory light-harvesting components for photosystems under iron limitation. In the present study, we investigated physiological features of Acaryochloris marina in response to iron-deficient conditions. The growth doubling time under iron-deficient conditions was prolonged to ~3.4 days compared with 1.9 days under normal culture conditions, accompanied with dramatically decreased chlorophyll content. The isolation of chlorophyll-binding protein complexes using sucrose density gradient centrifugation shows six main green bands and three main fluorescence components of 712, 728, and 748 nm from the iron-deficient culture. The fluorescence components of 712 and 728 nm co-exist in the samples collected from iron-deficient and iron-replete cultures and are attributed to Chl d-binding accessory chlorophyll-binding antenna proteins and also from photosystem II. A new chlorophyll-binding protein complex with its main fluorescence peak at 748 nm was observed and enriched in the heaviest fraction from the samples collected from the iron-deficient culture only. Combining western blotting analysis using antibodies of CP47 (PSII), PsaC (PSI) and IsiA and proteomic analysis on an excised protein band at ~37 kDa, the heaviest fraction (?F6) isolated from iron-deficient culture contained Chl d-bound PSI–IsiA supercomplexes. The PSII-antenna supercomplexes isolated from iron-replete conditions showed two fluorescence peaks of 712 and 728 nm, which can be assigned as 6-transmembrane helix chlorophyll-binding antenna and photosystem II fluorescence, respectively, which is supported by protein analysis of the fractions (F5 and F6).